The present invention relates to methods for determination of the presence of Bactericidal/permeability-increasing protein in a body fluid sample including a blood sample. Bactericidal/permeability-increasing protein (BPI) is a cationic anti-microbial protein which has been purified from the azurophilic granules of human and animal neutrophils (Weiss et al., J. Biol. Chem., 253:2664 (1978), Elsbach et al., J. Biol. Chem., 254:11000 (1979). BPI binds to the lipopolysaccharide (LPS) component of the outer membranes of gram-negative bacteria (Gazzano-Santoro et al., Infect. Immun., 60:4754 (1992)). Recently, a recombinant form of human BPI (rBPI23) has been characterized and compared to that of native BPI. The rBPI23 fragment consists of the amino-terminal 23 kcDa portion of holo-BPI and retains the LPS binding properties, as well as the anti-microbial activity, of native BPI (Gazzano-Santoro et al., J. Clin. Invest., 90:1122 (1992), Weiss, et al., J. Clin. Invest., 90:1122 (1992)).
BPI levels have not previously been accurately assayed in any body fluids. Because of the potential therapeutic use of rBPI23 and other BPI proteins and protein products, a sensitive and reproducible assay is needed to measure the presence and amount of BPI in body fluids. In particular, measurements of BPI in body fluids may be useful for diagnostic purposes. Pereira et al, J. Immunol. Methods, 117:115 (1989) discloses a competitive ELISA assay for the determination of BPI in crude granule extracts of human neutrophils. Pereira et al. also disclose that non-specific interactions of cationic proteins in an ELISA assay can be minimized by treatment with polyanions such as heparin or dextran sulfate. See also Pesce et al., J. Immunol. Methods, 87:21 (1986). However, the competitive assay of Pereira et al. is characterized by limited sensitivity. Accordingly, there remains a desire in the art for a more sensitive BPI assay capable of measuring endogenous BPI levels in mammalian body fluids. Also of interest to the present application is the disclosure of von der Mohien et al., Abstract, 13th International Symposium on Intensive Care and Emergency Medicine, Brussels (March 1993) disclosing the results of assays for serum levels of BPI in patients with gram-negative sepsis and healthy subjects. The abstract disclosed that no BPI was detectable under the conditions of the assay in the serum of healthy subjects while circulating BPI was detected in all septic patients.
The present invention provides methods for quantifying BPI levels in body fluid samples including blood samples according to the method of conducting a BPI immunoassay on a blood sample wherein the blood sample is plasma. Plasma is the blood fluid which remains after the white and red cells are separated from fresh uncoagulated blood. Serum is the blood fluid which remains when coagulated blood is separated by centrifugation (i.e., plasma without the blood clotting factors.) As one aspect of the invention it is taught that levels of BPI present in serum are not representative of endogenous levels of BPI in circulating blood while levels of BPI in plasma are. As a further aspect of the invention methods are provided for determining the presence of gram negative sepsis in a subject comprising determining the concentration of endogenous extracellular BPI in a plasma sample obtained from that subject and comparing that concentration with a standard value indicative of gram negative sepsis. Such a standard value can be 1.7 ng/mL which is two standard deviations above the mean value of 0.8 ng/mL for normal human plasma BPI. Values in excess of two standard deviations above the mean value for normal human plasma BPI concentrations are therefor indicative of gram negative sepsis.
Preferred methods according to the invention determine the concentration of extracellular BPI in body fluids such as blood plasma using a sandwich immunoassay and further utilize a cationic non-specific blocking agent selected from the group consisting of heparin and dextran sulfate in BPI immunoassays. The BPI immunoassays of the invention may also be used to determine the concentration of BPI in other body fluids including, but not limited to, serum, urine, lung lavages, vitreous fluid, crevicular fluid, cerebralspinal fluid, saliva and synovial fluid.
As a further aspect of the invention, methods for determining the presence of an active inflammatory state in a subject are provided which comprise determining the concentration of endogenous BPI in a fluid sample obtained from the subject and comparing that concentration with a standard indicative of an active inflammatory state. Where the body fluid being assayed to determine the presence of an active inflammatory state is blood plasma the standard indicative of an active inflammatory state can be 1.7 ng/mL which is two standard deviations above the mean value of 0.8 ng/mL for normal human plasma BPI. Values in excess of two standard deviations above the mean value for normal human plasma BPI concentrations are therefor indicative of the presence of an active inflammatory state. The invention further provides methods of determining the presence of active inflammatory states selected from the group consisting of rheumatoid arthritis and reactive arthritis by determining the concentration of endogenous BPI in a sample of synovial fluid obtained from the subject and comparing that concentration with a standard value indicative of an active inflammatory state. In the case of synovial fluid and rheumatoid arthritis and reactive arthritis the standard indicative of an active inflammatory state can be in excess of 152 ng/mL which is two standard deviations above the mean value of 26 ng/mL for synovial fluid BPI in a noninflammatory state.
Urine assays for BPI generally detect little or no BPI, although urine BPI levels may be elevated in subjects having urinary tract infections.